The Effects of Immunosuppressive Drugs on the Characteristics and Functional Properties of Bone Marrow-Derived Stem Cells Isolated from Patients with Diabetes Mellitus and Peripheral Arterial Disease

Abstract

Background: Diabetic patients (DPs) with foot ulcers can receive autologous cell therapy (ACT) as a last therapeutic option. Even DPs who have undergone organ transplantation and are using immunosuppressive (IS) drugs can be treated by ACT. The aim of our study was to analyze the effects of IS drugs on the characteristics of bone marrow-derived stem cells (BM-MSCs).

Methods: The cells were isolated from the bone marrow of DPs, cultivated for 14-18 days, and phenotypically characterized using flow cytometry. These precursor cells were cultured in the presence of various IS drugs. The impact of IS drugs on metabolic activity was measured using a WST-1 assay, and the expression of genes for immunoregulatory molecules was detected through RT-PCR. Cell death was analyzed through the use of flow cytometry, and the production of cytokines was determined by ELISA.

Results: The mononuclear fraction of cultured cells contained mesenchymal stem cells (CD45^-CD73⁺CD90⁺CD105⁺), myeloid angiogenic cells (CD45⁺CD146^-), and endothelial colony-forming cells (CD45^-CD146⁺). IS drugs inhibited metabolic activity, the expression of genes for immunoregulatory molecules, the production of cytokines, and the viability of the cells.

Conclusions: The results indicate that IS drugs in a dose-dependent manner had a negative impact on the properties of BM-MSCs used to treat ischemic diabetic foot ulcers, and that these drugs could affect the therapeutic potential of BM-MSCs.

Keywords: cell-based therapies; diabetes; diabetic foot ulcers; immunosuppressive drugs.

Figure 1

Characterization of BM-SC populations. (A) Representative dot plots show gating strategy of labeling live single-cell suspension analyzed by flow cytometry. (B) Representative histograms show that a single-cell suspension cultured for 14–18 days contained CD45⁻CD90⁺CD73⁺CD105⁺ cells (MSCs), and dot plot shows CD45⁺CD146⁻ cells (MACs and lymphocyte populations), CD45⁻CD146⁺ cells (ECFCs) and other cell populations.

Figure 2

The effect of IS drugs on the metabolic activity of BM-SCs. Cells were cultured for 48 h in the presence of tacrolimus, sirolimus, or mycophenolate mofetil, and metabolic activity was measured by WST-1 assay. Each bar represents the mean + SD measured in triplicate from 6 patients. Values with asterisks are significantly different from untreated control “0” (** p < 0.01; *** p < 0.001).

Figure 3

The effect of IS drugs on the expression of genes of immunoregulatory molecules in BM-SCs. Cells were cultured for 48 h in 3 different ways: (1) unstimulated (−), (2) stimulated with IFN-γ and TNF-α (+), or (3) stimulated with cytokines in the presence of sirolimus, tacrolimus, or mycophenolate mofetil. The expression of genes of immunoregulatory molecules IDO, iNOS, COX2, TGF-β, and PD-L1 was detected by RT-PCR. Each bar represents the mean + SD measured in triplicate from 6 patients. Values with asterisks are significantly different from stimulated control “+” (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

The impact of IS drugs on the production of cytokines and growth factors by BM-SCs. The cells were cultured in 3 different ways: (1) unstimulated (−), (2) stimulated with IFN-γ and TNF-α (+), or (3) stimulated with cytokines in the presence of various concentrations of tacrolimus, sirolimus, or mycophenolate mofetil. The production of cytokines by BM-SCs was determined in supernatants by ELISA. Each bar represents the mean + SD measured in triplicate from 6 patients. Values with asterisks are significantly different from stimulated control “+” (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

The effect of IS drugs on apoptosis (B) and cell death (C) of BM-SCs. The cells were cultured for 48 h untreated (0) or in the presence of different concentrations of tacrolimus, sirolimus, or mycophenolate mofetil, and the percentages of apoptotic and dead cells were measured by flow cytometry. Representative dot plots show the common gating strategy of labeling single-cell suspension, control (Anexin V⁻ Hoechst3358⁻), and both groups of cultured BM-SCs: untreated and exposed to mycophenolate mofetil (50 µg/mL) (A). Each bar represents the mean + SD measured in triplicate from 6 patients. Values with asterisks are significantly different from control “0” (** p < 0.01; *** p < 0.001).